Two-dimensional (2D) cultures of cancer cells are routinely used in drug discovery for screening and initial characterization of the efficacy of librry of potential drug compounds. Despite their simplicity and compatibility with high throughput screening instruments, 2D cell assays often fail to predict the efficacy of compounds in vivo, making drug development and discovery an extremely costly process. Disparity between 2D cultures and the complex 3D environment of cancer cells in vivo is the major shortcoming of monolayer culture systems. Development of novel chemotherapeutics requires compound screening against malignant tumor cells in a setting that resembles the 3D tumor environment. Cancer cell spheroids (CCS) are 3D clusters of malignant cells that are regarded as physiologic models of solid tumors; they possess similar metabolic and proliferative gradients to avascular tumors and exhibit the clinical expression profiles of solid tumors. Despite the inherent power of CCS to predict clinical efficacy of drugs, incorporation of CCS into the mainstream drug development process is severely hindered by complex and expensive methodological requirements for the formation and maintenance of CCS. We overcome the limitations of existing techniques by developing a technological platform to generate spheroids of consistent size in standard 384-microwell plates using an aqueous two- phase system (ATPS). A drop of the denser aqueous phase containing cancer cells is robotically dispensed into each well containing the second, immersion phase. The drop confines cells and remains immiscible from the immersion phase to facilitate aggregation of cells into a compact CCS of well-defined size. Importantly the overlay of culture media provides nutrients and minimizes the well-known problem of evaporation and changes in osmolality of media as in other assays. The entire process of generating 384 spheroids is done robotically and in a single step. The unprecedented ease of formation and maintenance of CCS and full compatibility with standard equipment in high throughput screening laboratories makes this microtechnology readily available to the researchers in academia and industry. We anticipate that this microtechnology will make drug testing and screening with 3D tumor models a routine laboratory technique prior to expensive and tedious in vivo analyses. In addition, it will dramatically improve testing throughput and cost-effectiveness (increasing numbers of tested compounds and reduced reagent consumption) and efficiency (reducing hands-on time) to expedite drug discovery. We will accomplish our goals through two specific aims: (i) Generation of cancer cell spheroids with aqueous biphasic systems; (ii) Initial validation of ATPS spheroids for compound testing.